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2.
Curr Protoc ; 2(1): e340, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35007410

RESUMO

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.


Assuntos
Calcificação de Dente , Dente , Animais , Durapatita , Camundongos , Microscopia Eletrônica de Varredura , Microtomografia por Raio-X
4.
Front Cell Dev Biol ; 9: 620692, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34150743

RESUMO

WIZ (Widely Interspaced Zinc Finger) is associated with the G9a-GLP protein complex, a key H3K9 methyltransferase suggesting a role in transcriptional repression. However, its role in embryonic development is poorly described. In order to assess the loss of function of WIZ, we generated CRISPR/Cas9 WIZ knockout mouse model with 32 nucleotide deletion. Observing the lethality status, we identified the WIZ knockouts to be subviable during embryonic development and non-viable after birth. Morphology of developing embryo was analyzed at E14.5 and E18.5 and our findings were supported by microCT scans. Wiz KO showed improper development in multiple aspects, specifically in the craniofacial area. In particular, shorter snout, cleft palate, and cleft eyelids were present in mutant embryos. Palatal shelves were hypomorphic and though elevated to a horizontal position on top of the tongue, they failed to make contact and fuse. By comparison of proliferation pattern and histone methylation in developing palatal shelves we brought new evidence of importance WIZ dependent G9a-GLP methylation complex in craniofacial development, especially in palate shelf fusion.

5.
Development ; 148(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34032267

RESUMO

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.


Assuntos
Plexo Corióideo/embriologia , Epitélio/metabolismo , Quarto Ventrículo/embriologia , Proteína Meis1/metabolismo , Proteína Wnt-5a/metabolismo , Animais , Encéfalo/embriologia , Sistemas CRISPR-Cas/genética , Linhagem Celular , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de Sinais/fisiologia , Proteína Wnt-5a/genética
6.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916973

RESUMO

NME7 (non-metastatic cells 7, nucleoside diphosphate kinase 7) is a member of a gene family with a profound effect on health/disease status. NME7 is an established member of the ciliome and contributes to the regulation of the microtubule-organizing center. We aimed to create a rat model to further investigate the phenotypic consequences of Nme7 gene deletion. The CRISPR/Cas9 nuclease system was used for the generation of Sprague Dawley Nme7 knock-out rats targeting the exon 4 of the Nme7 gene. We found the homozygous Nme7 gene deletion to be semi-lethal, as the majority of SDNme7-/- pups died prior to weaning. The most prominent phenotypes in surviving SDNme7-/- animals were hydrocephalus, situs inversus totalis, postnatal growth retardation, and sterility of both sexes. Thinning of the neocortex was histologically evident at 13.5 day of gestation, dilation of all ventricles was detected at birth, and an external sign of hydrocephalus, i.e., doming of the skull, was usually apparent at 2 weeks of age. Heterozygous SDNme7+/- rats developed normally; we did not detect any symptoms of primary ciliary dyskinesia. The transcriptomic profile of liver and lungs corroborated the histological findings, revealing defects in cell function and viability. In summary, the knock-out of the rat Nme7 gene resulted in a range of conditions consistent with the presentation of primary ciliary dyskinesia, supporting the previously implicated role of the centrosomally located Nme7 gene in ciliogenesis and control of ciliary transport.


Assuntos
Transtornos da Motilidade Ciliar/genética , Genes Letais , Predisposição Genética para Doença , Núcleosídeo-Difosfato Quinase/deficiência , Animais , Cílios/metabolismo , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/diagnóstico , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Genótipo , Imuno-Histoquímica , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fenótipo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Transcriptoma , Microtomografia por Raio-X
7.
Nature ; 593(7859): 424-428, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33767445

RESUMO

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , COVID-19/virologia , Imunoglobulina G/imunologia , SARS-CoV-2/imunologia , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/uso terapêutico , Peso Corporal , COVID-19/prevenção & controle , Dependovirus/genética , Modelos Animais de Doenças , Epitopos de Linfócito B/química , Epitopos de Linfócito B/imunologia , Feminino , Humanos , Evasão da Resposta Imune/genética , Camundongos , Camundongos Endogâmicos C57BL , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Tratamento Farmacológico da COVID-19
8.
bioRxiv ; 2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33501434

RESUMO

Neutralizing antibodies targeting the receptor binding domain (RBD) of the SARS-CoV-2 Spike (S) are among the most promising approaches against coronavirus disease 2019 (COVID-19) 1,2 . We developed a bispecific, IgG1-like molecule (CoV-X2) based on two antibodies derived from COVID-19 convalescent donors, C121 and C135 3 . CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable S binding to Angiotensin-Converting Enzyme 2 (ACE2), the virus cellular receptor. Furthermore, CoV-X2 neutralizes SARS-CoV-2 and its variants of concern, as well as the escape mutants generated by the parental monoclonals. In a novel animal model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, combining into a single molecule the advantages of antibody cocktails.

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